Chemical analysis of fluids, including body fluids such as blood, serum, urine, and so forth; water, fluid foodstuffs, etc., is often desirable and frequently necessary. Safety concerns, medical diagnosis, forensics, and other fields rely on determinations, either qualitative or quantitative, of components of fluids. These determinations, or assays, must be rapid and accurate.
A major field of clinical analysis is devoted to "dry chemistry" determinations of components in liquid samples. "Dry chemistry" refers to the apparatus used in the determinations, because the apparatus is dry to the touch. Generally, these apparatus take the form of monolayer and multilayer test strips and analytical test elements. These analytical devices have excellent storage and handling properties, and are convenient to use.
The determination of an analyte in a sample typically involves reacting the analyte with a binding partner which undergoes some type of change following reaction, leading to a detectable signal. While the change may be caused directly by the reaction with the analyte, frequently this is not the case, as the change usually results from some property produced by the interaction between analyte and reaction product which does not exist in unreacted components.
Several types of assay have been developed which utilize the above discussed principles. One important type is the immunoenzymometric assay. This test involves the binding of the analyte being tested with a reaction partner which carries a label. The binding partner is contained in a test strip or other apparatus in such a way that it is non-reactive unless and until its partner analyte contacts the test strip, in the form of a fluid sample. When this happens, the analyte and labeled binding partner bind to each other to form a complex, which must then be determined. This is accomplished by reacting the label carried by the binding partner with another substance so as to form a detectable signal. When the label is an enzyme, as it frequently is, the substance used is a substrate for the enzyme which, upon reaction with the enzyme, either forms a visible color or changes color. Measuring the change or amount of color gives a measure of complex, and hence of analyte.
A problem with this system, is that one must, have sufficient labeled binding partner to bind essentially all of analyte in the sample. The amount of analyte, however, is not known. This thus makes it necessary to provide excess amounts of labeled binding partner. Some of this will not react with analyte, but as it carries the label, it nonetheless forms a detectable signal. Thus, unless one separates reacted label carrier from the unreacted portion, no test result can be achieved.
Separation does, however, take place. An immunoenzymometric assay has an additional feature in that after the sample has contacted the labeled binding partner and some of the latter has been bound to form the complex, the mixture of complex and unreacted labeled binding partner is contacted to a sample of solid phase bound analyte or an analyte analogue which binds to uncomplexed labeled binding partner, and removes it from the solution mixture. One then has a clean division of labeled partner bound to analyte, and excess labeled binding partner bound to solid phase. Addition of substrate to either of these gives a color. If the amount of the labeled moiety with which one began is known, one measures the amount of signal either in the liquid phase, which gives a direct relationship to the amount of analyte, or in the solid phase, which provides an indirect relationship.
This type of assay involves two phases, and is thus called a heterogeneous assay. When performed using a test strip, one has the advantage of having labeled moiety, immobilized component, and reactive substance for the label all in one device. Fluid samples will diffuse through test strips, and thus the reactions will occur quickly and without the need for intervention by the investigator.
The immunoenzymometric assay is not the only type of assay used for these analytical systems. A competitive assay uses, rather than labeled binding partner, a sample of labeled analyte corresponding to the analyte being determined. The solid phase bound reactant, in these cases, is the binding partner for the analyte and labeled analyte. If any of the analyte is in the sample, competition for the binding sites ensues. One then measures the amount of label either in the solid phase or the liquid phase in the same manner described for immunoenzymometric assays, to determine the analyte.
Yet another system, which bears some relationship to the competitive assay, is the displacement assay. In this system, labeled analyte is already bound to a solid phase. When the sample contacts the test strip containing the bound labeled analyte, some of the label will be displaced by the binding between sample analyte and solid phase bound binding partner, and again, measurement in one of the phases is carried out.
Still another system which may be mentioned is a sandwich assay. Sandwich assays encompass a broad range of types of assays. For the invention described herein, however, a sandwich assay refers to formation of a complex between the analyte An being determined, a labeled, epitopically active fragment Fab.sup.* of a monoclonal antibody, and a nonlabeled whole monoclonal antibody mAb. The sandwich which forms when analyte contacts a test strip containing diffusible mAb and Fab.sup.* is mAb-An-Fab.sup.*. This sandwich contacts a solid phase containing another antibody, which binds to mAb, but not to Fab.sup.*. The result of this is to separate complex from uncomplexed Fab.sup.*, so as to permit determination in the same manner discussed supra for other systems. The prior art, which is discussed infra gives many examples of different forms of these systems.